EP1474522B1 - Methode de generation de cellules genetiquement modifiees pour la regulation et l'analyse de genes specifiques d'un locus - Google Patents

Methode de generation de cellules genetiquement modifiees pour la regulation et l'analyse de genes specifiques d'un locus Download PDF

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EP1474522B1
EP1474522B1 EP03703842A EP03703842A EP1474522B1 EP 1474522 B1 EP1474522 B1 EP 1474522B1 EP 03703842 A EP03703842 A EP 03703842A EP 03703842 A EP03703842 A EP 03703842A EP 1474522 B1 EP1474522 B1 EP 1474522B1
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cell
gene
locus
cells
specific targeting
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EP1474522A4 (fr
EP1474522A1 (fr
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Luigi Grasso
J. Bradford Kline
Nicholas C. Nicolaides
Philip M. Sass
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Morphotek Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells

Definitions

  • the invention is related to the area of homologous recombination in eukaryotic cells for studying gene function, gene expression, and generating over-producer clones for high protein production. In particular it is related to the field of therapeutic target discovery, pharmacologic compound screening and protein manufacturing.
  • the use of specific gene targeting in eukaryotic cell-based model systems provides an effective and selective strategy for studying the function of a particular gene in response to biological or chemical molecules as well as for model systems to produce biochemicals for therapeutic use.
  • homologous recombination to: (1) inactivate gene function to study downstream functions; (2) introduce reporter gene molecules into targeted loci to facilitate the screening of gene expression in response to biomolecules and/or pharmaceutical compounds; (3) generate stable, steady-state expression of target genes via the introduction of constitutively active heterologous promoter elements or through chromosomal site-specific gene amplification.
  • WO01l/68882 discloses a method of promoting an alternation at a selected site in a target DNA, including providing a double-stranded DNA sequence which includes a selected DNA sequence, an agent which enhances homologous recombination and an agent which inhibits non-homologous end joining.
  • US5272071 disclose a process for the modification of the expression characteristics of a gene normally present within the genome of a stable cell line or cloned microorganism by inserting an appropriate regulatory segment into the gene by homologous recombination.
  • US5922601 discloses a gene trap construct for identification of genes whose activity is regulated upon a cellular transition event which comprises, in downstream sequence, (1) a cassette having a functional or splice acceptor, a translation stop sequence and an internal ribosome entry site, and (2) a promoterless protein coding sequence encoding at least one polypeptide providing positive and negative selections traits.
  • US6166178 discloses compositions and methods related to telomerase reverse transcriptase.
  • US6146894 discloses the use of dominant negative alleles of human mismatch repair genes to generate hypermutable cells and organisms.
  • the ability to generate site-directed "knock-ins" in eukaryotic cells, in particular mammalian cells, used for drug screening or development of custom cell lines for constitutive gene expression is of great value for pharmaceutical drug product development as well as for compound screening.
  • Compounds can be of a low molecular weight, a complex macromolecule or protein.
  • the compound can be targeted to a gene of interest whose expression is altered either positively or negatively by directly or indirectly affecting the activity of promoter and/or enhancer elements that are involved in regulating the expression of a specific gene locus.
  • One method taught in this application is the "knock-in" of constitutively active promoter elements (such as but not limited to viral promoters, i.e .
  • SV40 early or late promoters CMV, LTR, etc. or promoters from constitutively expressed housekeeping genes such as the elongation factor or actin) into a desired locus.
  • the ability to direct constitutive gene expression from a host organisms genome may lead to the establishment of cell lines such as but not limited to those that overproduce therapeutic targets for drug binding studies, gene function studies as well as lines that overproduce therapeutic proteins for product manufacturing applications.
  • DHFR dihydrofolate reductase
  • the invention provides an in vitro method of introducing a locus specific targeting fragment into the genome of a cell through homologous recombination comprising:
  • the invention also provides an in vitro method of genetically altering a cell to overproduce a selected polypeptide comprising:
  • the invention also provides an in vitro method of tagging an exon of a cell for screening gene expression in response to biochemical or pharmaceutical compounds comprising:
  • the invention also provides an in vitro method of tagging a specific chromosomal site for locus-specific gene amplification comprising:
  • the method further comprises restoring mismatch repair activity of the cell.
  • the promoter may be a CMV promoter, an SV40 promoter, elongation factor, LTR sequence, a pIND promoter sequence, a tetracycline promoter sequence, or a MMTV promoter sequence.
  • the selectable marker may be a hygromycin resistance gene, a neomycin resistance gene or a zeocin resistance gene.
  • the 5' and 3' flanking regions are 30 to 100 nucleotides in length. In other embodiments of the methods of the invention, the 5' and 3' flanking regions are 40 to 90 nucleotides in length. In other embodiments of the methods of the invention, the 5' and 3' flanking regions are 50 to 80 nucleotides in length. In other embodiments of the methods of the invention, the 5' and 3' flanking regions are 50 to 70 nucleotides in length.
  • the cell may be a vertebrate cell, an invertebrate cell, a mammalian cell, a reptilian cell, a fungal cell, or a yeast cell.
  • the 5' and 3' flanking regions are homologous to a 5' flanking region of a selected chromosomal locus of the cell.
  • the reporter element may be a form of luciferase or a green fluorescent protein.
  • the reporter element is fused in frame to the selectable marker.
  • the locus specific targeting fragment may further comprise a selectable marker and a second promoter operatively linked to the selectable marker.
  • homologous recombination of small overlapping DNA regions is difficult to achieve, however, it is taught by this application that the use of inhibiting mismatch repair (MMR) in eukaryotic somatic cells increases the efficiency of homologous recombination that allows for the rapid generation of recombination using homologous regions as short as 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 nucleotides in length.
  • the homologous regions are as short as 25 to 115 nucleotides in length. In other embodiments, the homologous regions are as short as 30 to 110 nucleotides in length.
  • the homologous regions are as short as 35 to 105 nucleotides in length. In other embodiments, the homologous regions are as short as 40 to 100 nucleotides in length. In other embodiments, the homologous regions are as short as 45 to 95 nucleotides in length. In other embodiments, the homologous regions are as short as 50 to 90 nucleotides in length. In other embodiments, the homologous regions are 50 to 85 nucleotides in length. In other embodiments, the homologous regions are 50 to 80 nucleotides in length. In other embodiments, the homologous regions are 50 to 75 nucleotides in length. In other embodiments, the homologous regions are 50 to 70 nucleotides in length.
  • MMR MMR-induced mutator phenotype
  • Nicolaides, N.C. et al. (1998) "A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype" Mol. Cell. Biol. 18:1635-1641 ; U.S. Patent No. 6,146,894 to Nicolaides et al .).
  • MMR is restored by removal of the dominant negative allele and hosts are selected for integrated fragments by selection of the appropriate marker gene.
  • somatic eukaryotic cells containing knocked-in expression control elements or exon-tags, or DHFR amplification units will facilitate studies on elucidating unknown gene function by the ability to over express genomic loci at will under a variety of experimental growth conditions in the presence or absence of exogenous biological or pharmacological factors.
  • the use of such an approach to specifically tag a gene's exon will facilitate the profile of gene expression under certain growth conditions in wild type and pathogenic cells grown in the presence or absence of biological or pharmaceutical factors.
  • the ability to specifically amplify chromosomal regions can facilitate enhanced protein production in a given host organism for discovery, development, and/or manufacturing or a given gene product.
  • the invention described herein is directed to the creation of genetically modified eukaryotic cells, in particular, somatic mammalian cells containing targeted loci with regulated or constitutively active expression elements for the use in uncovering gene function or polypeptide production as well as the use of targeting vectors that can tag an exon of a locus which can subsequently be monitored in response to biological or pharmaceutical molecules.
  • the ability to generate such cells are facilitated by the use of targeting cassettes containing elements that are rapidly modified to target a given locus via PCR-mediated synthesis using locus specific primers containing 20-120 nts, specifically 50-70 nts, of homologous sequence to the chromosomal target site in combination with the use of agents that can block the endogenous MMR of the host during DNA integration to increase recombination efficiency of short homologous sequences (Nicholas Nicolaides, personal observation).
  • the present invention describes the facilitated synthesis of gene targeting fragments for controlling gene expression from the chromosomal site within eukaryotic cells as well as the use of exon-tagging fragments to study gene expression in the presence of biological or pharmaceutical agents.
  • the advantages of the present invention are further described in the examples and figures described herein.
  • the invention provides methods for generating genetically altered cell lines that overproduce polypeptides for function studies. In other embodiments, the invention provides methods for generating genetically altered cell lines that overproduce polypeptides for production purposes. In other embodiments, the invention provides methods for generating genetically altered cell lines with genes whose exons are tagged for screening purposes.
  • the invention provides methods of enhancing the frequency of homologous recombination of a DNA fragment within a specific chromosomal locus in eukaryotic cells by blocking the MAR activity of the somatic cell host.
  • the invention provides methods of creating targeted eukaryotic cell lines with chromosomal loci containing DHFR expression vector for locus-specific gene amplification.
  • a method for making a somatic eukaryotic cell line MMR defective, followed by the introduction of a locus specific targeting fragment that results in the constitutive expression of a chromosomal locus is provided.
  • a polynucleotide encoding a dominant negative allele of a MMR gene comprising PMS2-134 is introduced into a target cell.
  • the cell becomes hypermutable as a result of the introduction of the gene.
  • a targeting fragment is generated by PCR using primers containing sequences homologous to the chromosomal locus of interest.
  • the fragment is introduced into the host by transfection. Cell pools are then selected for clones with integrated fragments.
  • Selected clones are further analyzed by any number of means to assess expression and/or genome integration of a specific site. Upon confirmation of site-desired integration, MMR is restored in clones and the cells are useful for functional studies or for generating high levels of protein for product development and/or manufacturing applications.
  • a cell line with a targeted exon is provided.
  • a somatic eukaryotic cell line is rendered MMR defective by introduction of a dominant negative MMR gene allele comprising PMS2-134, followed by the introduction of a targeting fragment containing a reporter gene fused to a selectable marker that results in the tagging of an endogenous gene's exon is provided.
  • a polynucleotide encoding a dominant negative allele of a MMR gene comprising PMS2-134 is introduced into a target cell. The cell becomes hypermutable as a result of the introduction of the gene.
  • a targeting fragment is generated by PCR using primers containing sequences homologous to the chromosomal locus of interest. The fragment is introduced into the host by transfection.
  • Cell pools are then selected for clones with integrated fragments. Selected clones are further analyzed by any number of means to assess expression and/or genome integration of a specific site. Upon confirmation of site-desired integration, MMR is restored in clones and the cells are useful for fimctional studies to profile endogenous gene expression in the presence or absence of biological or pharmacological factors.
  • a cell line with a targeted locus is provided.
  • a somatic eukaryotic cell line is rendered MMR defective by introduction of a dominant negative MMR gene allele comprising PMS2-134, followed by the introduction of a targeting fragment containing a DHFR gene and a selectable marker that results in the specific tagging of a chromosomal site is described.
  • a polynucleotide encoding a dominant negative allele of a MMR gene comprising PMS2-134 is introduced into a target cell. The cell becomes hypermutable as a result of the introduction of the gene.
  • a targeting fragment is generated by PER using primers containing sequences homologous to the chromosomal locus of interest.
  • the fragment is introduced into the host by transfection.
  • Cell pools are then selected for clones with integrated fragments. Selected clones are further analyzed by any number of means to assess expression and/or genome integration of a specific site.
  • cells are selected for methotrexate (MTX) resistance.
  • MTX-resistant cells are then analyzed for chromosomal site amplification using any means useful to those skilled in the art such as but not limited to genomic analysis by southern blot, RNA expression analysis or protein expression analysis.
  • MMR is restored in clones and the cells are useful for functional studies to profile endogenous gene expression in the presence or absence of biological or pharmacological factors as well as for production strains.
  • inventions provide the art with methods that can rapidly generate gene targeted eukaryotic cells whereby the locus of interest can have altered expression profiles to study gene function and/or enhanced production levels for manufacturing. Moreover, the invention provides the art with methods to tag an exon of a gene that is useful for monitoring gene expression within a given host.
  • Figure 1 shows a schematic diagram of promoter locus-specific targeting fragments (LSTF) and the genomic organization of a target gene.
  • Primer Set A indicates the primer position of the oligonucleotides used to generate the LSTF for each gene that is useful for genome analysis.
  • Primer Set B indicates the primer position of oligonucleotides used to analyze each target gene to confirm locus specific integration.
  • the box below each gene represents the LSTF, where the shaded areas represent the areas of homology to the target gene, whereby the homologous region is 50-70 nts in length.
  • the black boxes in the gene diagram represents exons that are numbered with respect to homology to the target gene whereby sensitive RT-PCR can be used to assay for fusion spliced cDNAs consisting of CMV leader sequence located 3' to the CMV promoter elements.
  • the targeting cassette is used for generating constitutive expression from a eukaryotic host's genome.
  • Figure 2 shows expression of B-globin in HEK293 cells transfected with LSTFs.
  • Reverse transcriptase PCR was carried out using equal amounts of total RNA from each cell line and a 5' primer located in the leader sequence downstream of the CMV promoter (SEQ ID NO:21) and a 3' primer located in the coding region of the beta-globin gene (SEQ ID NO:25).
  • PCR reactions were electrophoresed on 2% agarose gels, ethidium bromide stained and visualized using a UV light box.
  • the arrow indicates a product of the expected molecular weight.
  • Figure 3A shows the sequence of the fusion gene hygromycin-green fluorescence binding protein for exon tagging of somatic cells.
  • the sequence in bold encodes for the hygromycin resistance gene, while the sequence in normal font encodes the green fluorescence binding protein.
  • Figure 3B shows the sequence of the fusion gene hygromycin-luciferase for exon tagging of somatic cells.
  • the sequence in bold encodes for the hygromycin resistance gene, while the sequence in normal font encodes the luciferase protein.
  • FIG. 4 shows a schematic diagram of exon locus-specific targeting fragments (LSTF) and the genomic organization of a target gene.
  • the LSTF contains a selectable marker gene (i.e ., hygromycin, neomycin, zeocin, etc .) that is in frame with a reporter gene, (i.e ., luciferase, Green Fluorescent Protein, etc .).
  • Primer Set A indicates the primer position of oligonucleotides used to analyze each target gene to confirm locus specific integration where the 5' primer is located in the exon preceding the targeted exon and the 3' primer is located proximal to the site of integration.
  • each gene represents the LSTF, where the shaded areas represent the areas of homology to the target gene, whereby the homologous region is 50-70 nts in length.
  • the black boxes in the gene diagrams represent exons whereby RT-PCR can be used to assay for fusion of spliced cDNAs consisting of the selectable marker-reporter cDNA within the targeted gene's encoded transcript.
  • MMR mismatch repair
  • inhibitor of mismatch repair refers to an agent that interferes with at least one function of the mismatch repair system of a cell and thereby renders the cell more susceptible to mutation.
  • hypomutable refers to a state in which a cell in vitro is made more susceptible to mutation through a loss or impairment of the mismatch repair system.
  • agents when used in connection with inhibition of MMR refers to chemicals, oligonucleotides, analogs of natural substrates, and the like that interfere with normal function of MMR.
  • gene is used herein to denote a DNA segment encoding a polypeptide, and includes genomic DNA (with or without intervening sequences), cDNA, and synthetic DNA. Genes may include non-coding sequences, including promoter elements.
  • operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g ., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • promoter elements are used to denote sequences within promoters that function in the initiation of transcription and which are often characterized by consensus nucleotide sequences.
  • Promoter elements include RNA polymerase binding sites; TATA sequences; CAAT sequences; differentiation-specific elements (DSEs; McGehee et al. (1993) Mol. Endocrinol. 7:551-560 ; cyclic AMP response elements (CREs); serum response elements (SREs; Treisman (1990) Seminars in Cancer BioL 1:47-58 ); glucocorticoid response elements (GREs); and binding sites for other transcription factors, such as CRE/ATF ( O'Reilly et al. (1992) J.
  • Transcription regulatory elements are promoter-associated DNA sequences that bind regulatory molecules, resulting in the modulation of the frequency with which transcription is initiated. Transcription regulatory elements can be classified as enhancers or suppressors of transcription.
  • reporter gene is used herein to denote a gene that, when expressed in a cell, produces a quantifiable phenotypic change in the cell.
  • Preferred reporter genes include genes encoding enzymes. Particularly preferred enzymes are luciferase, ⁇ -galactosidase, and chloramphenicol acetyltransferase. Assays for these enzymes are known in the art. See, for example, Seed and Sheen (1988) Gene 67:271-277 ; Todaka et al. (1994) J. Biol Chem. 269:29265-29270 ; Guarente et al. (1981) Proc. Natl. Acad Sci.
  • the inventors have discovered a method for developing a rapid method for knocking in DNA fragments into target loci of interest to regulate gene expression and/or function as well as the ability to rapidly tag an exon of a gene to study expression as well as for enhancing chromosomal site-specific gene amplification.
  • the process entails the use of targeting cassettes that are generated via PCR using primers containing 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 nucleotides of sequence with homology to a particular chromosomal locus.
  • Each promoter expression cassette contains DNA elements that can produce constitutive-, inducible- or suppressed-expression, which are juxtaposed to a constitutively expressed selectable marker (See Fig. 1 ).
  • Each exon-tag cassette contains DNA sequences encoding for reporter elements that can be monitored using a number of detection methods such as but not limited to green fluorescent protein, luciferase, etc., which is fused in-frame to a selectable marker (See Fig. 4 ).
  • Each DHFR expression cassette contains DNA elements that constitutively express DHFR which are juxtaposed to a constitutively active selectable marker. In all cases, targeting fragments are generated and transfected into eukaryotic cell hosts.
  • Enhanced site-specific homologous recombination of LSTFs is facilitated in each target cell by suppressing the endogenous MMR of the host via the expression of a dominant negative MMR gene mutants comprising PMS2-134 as described ( Nicolaides, N.C. et al. (1998) "A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype” Mol. Cell. Biol 18:1635-1641 ; U.S. Patent No. 6,146,894 to Nicolaides et al .; Lipkin et al. (2000) "Na,H3: a DNA mismatch repair gene associated with mammalian microsatellite instability" Nat. Genet. 24:27-35 ).
  • the methods taught here are useful for the generation of cells that over express or suppress the expression of a gene(s) to elucidate gene function.
  • Such cells may be used as tools to identify compounds that can alter the activity of a given gene product and/or induced pathway in comparison to parental lines.
  • the cell host may be derived from a variety of sources, for example, normal or pathogenic tissues or organisms.
  • the targeting fragment may be used, for example, to prevent, inhibit or terminate expression of a particular gene to elucidate its function, if any, in a particular disease-associated pathway.
  • such cell lines may now be used to screen compound libraries to identify molecules that act as agonists or antagonists for pharmaceutical product development.
  • GCR G coupled receptors
  • the methods are useful for the generation of cells with endogenous genes containing a tagged exon for monitoring gene expression profiles.
  • Such cells may be used as tools to monitor physiological activity in the presence or absence of exogenous factors in comparison to control lines.
  • the cell host may be derived from, for example, normal or pathogenic organisms to study the expression profile of disease associated genes under normal or stimulated conditions. Pharmacological studies can be performed in untreated cultures or in cultures treated with biological or chemical factors to screen for therapeutic molecules.
  • the cell lines produced by the method of the invention containing tagged exons are also useful for monitoring compound toxicity and efficacy of modulating gene expression.
  • Reporter elements may be included in the constructs of the invention. Reporter elements include assayable proteins which can be detected and/or quantified. Examples of reporter genes include, but are not limited to luciferases, such as those known in the art, and may include firefly luciferase (amino acid, SEQ ID NO:34, nucleic acid SEQ ID NO:35); bacterial luciferases, and Renilla luciferase (amino acid, SEQ ID NO:38, nucleic acid SEQ ID NO:39) and green fluorecence protein (amino acid, SEQ ID NO:36, nucleic acid SEQ ID NO:37). Other reporter elements include genes encoding enzymes, which convert a substrate that is subsequently detected. Examples include, but are not limited to ⁇ -galactosidase, and chloramphenicol acetyl transferase.
  • the reporter gene may be visualized in a variety of assays including both in vivo and in vitro assays.
  • reporter genes can be visualized by positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and flurorescence with wild-type and mutant green fluorescent protein and luciferase (see Ray et al. (2001) "Monitoring gene therapy with reporter gene imaging” Semin. Nucl. Med. 31(4):312-320 ).
  • Renilla luciferase reporter gene could be used and detected to follow gene expression in vivo ( Bhaumik and Gambhir (2002) Proc. Natl. Acad. Sci. USA 99(1):377-382 ).
  • a highly sensitive cooled charge-coupled device (CCD) camera provided images of photon counting.
  • CCD charge-coupled device
  • Such a device is suitable for use in the present invention, and is available from Xenogen ( In Vivo Imaging System "IVIS").
  • IVIS In Vivo Imaging System
  • a description of the protocols used to image the reporter gene is known in the art ( Bhaumik and Gambhir (2002) Proc. Natl. Acad. Sci. USA 99(1):377-382 ) and are suitable for use in the present invention as assays to monitor expression of reporter genes.
  • a bifunctional molecule comprising Renilla luciferase and Green Fluorescent Protein may be used as a reporter gene to monitor the integration and/or expression of the LSTF construct.
  • a ruc-gfp fusion gene construct was created by fusing cDNAs for Renilla luciferase (ruc) and "humanized" GFP (gfp) from Aequorea in frame, and the construct was subsequently expressed in mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of the substrate, coelenterazine, and GFP fluorescence upon excitation with UV light.
  • proteins expressed from LSTFs may be visualized in vitro using labeled antibodies, or fragments thereof (such as Fab or F(ab')2 fragments) which specifically bind to the protein of interest.
  • Antibodies may be labeled using any means known in the art that allow visualization or assaying.
  • labels include, but are not limited to fluorescent conjugates, and radioactive conjugates.
  • Fluorescent conjugates include luciferases, green fluorescent protein and derivatives, rhodamine, and fluorescein.
  • Radioactive compounds include those containing 131 I, 111 I, 123 I, 99 mTC, 32 P, 125 I, 3 H, and 14 C.
  • the antibody or fragments thereof can be labeled with such reagents using techniques known in the art (see, for example, Wensel and Meares, Radioimmunoimaging and Radioimmunotherapy, Esevier, New York (1983 ); D. Colcher et al. (1986) "Use of Monoclonal Antibodies as Radiopharmaceuticals for the Localization of Human Carcinoma Xenografts in Athymic Mice" Meth. Enzymol. 121:802-816 ).
  • signaling mechanisms that may be affected by proteins expressed by LSTFs may be monitored or assayed for functionality.
  • calcium flux may be measured in cells expressing receptors that affect calcium flux upon stimulation.
  • protocols that measure calcium mobilization are the FLIPR ® Calcium Assay Kit, and various protocols using the calcium binding, fluorescent dye, Fluo-3 AM. The protocols are known to those of skill in the art and may be used to measure calcium mobilization in cells expressing various proteins (such as G-protein coupled receptors, for example) which have been expressed from an LSTF.
  • the LSTF for use in the invention may be constructed to include a variety of genetic elements, depending on the application of the LSTF.
  • a LSTF may include a promoter operatively linked to a selectable marker.
  • the LSTF may include a promoter operatively linked to a selectable marker and a second protein encoding sequence operatively linked to a second promoter.
  • an internal ribosome entry site IRES
  • An IRES element is a regulatory element found in some viral sequences and some cellular RNAs that enhances translation of a second gene product in a bicistronic eukaryotic expression cassette ( Kaufman et al.
  • An IRES element may be engineered between two of the coding sequences of the LSTFs of the invention.
  • a promoter is not required.
  • it is sufficient that a nucleic acid sequence is present on the construct which may be detectable through molecular analysis.
  • constructs include a promoter operatively linked to a dihydrofolate reductase encoding sequence, preferably with a second promoter operatively linked to a selectable marker.
  • a selectable marker may be a gene conferring drug-resistance to the cell.
  • drug resistance selectable markers are genes for neomycin resistance, hygromycin resistance and zeocin resistance.
  • a locus control region may be incorporated.
  • An LCR is position and orientation dependent and may be used in a tissue specific manner.
  • An LCR may be used in the LSTF of the invention in conjunction with a promoter in embodiments used for overproduction of protein.
  • an PCR specific for lymphocytes may be used to produce high levels of antibodies in B cells using LSTFs that integrate through homologous recombination in the immunoglobulin locus. LCRs are known by persons skilled in the art.
  • the constructs are amplified in a polymerase chain reaction (PCR) using 5' and 3' primers that have been designed to include nucleic acid sequence that is homologous to a selected portion of the genome of a cell that is targeted for homologous recombination.
  • PCR polymerase chain reaction
  • the 5' primer which anneals to the (-) strand of the DNA in the PCR amplification
  • the 5'-most sequence of the 5' primer (about 20-120 nucleotides (nts)) is homologous to the selected portion of the genome targeted for homologous recombination.
  • the 3' most portion of the 5' primer comprises nucleotides that are homologous to the 5' portion of the construct to be amplified.
  • the 5'-most sequence of about 20-120 nucleotides (nts) is homologous to the selected portion of the genome targeted for homologous recombination.
  • the 3' most portion of the 3' primer comprises nucleotides that are homologous to the 3' portion of the construct to be amplified.
  • the PCR reaction conditions are not particularly limited. PCR reactions and variations for optimization are well known in the are and routine optimization of the reactions, including choice of buffers, polymerases, additives, etc., are in the purview of the skilled artisan.
  • a polynucleotide encoding for a dominant negative form of a MMR protein comprising PMS2-134 is introduced into a cell.
  • the polynucleotide can be in the form of genomic DNA, cDNA, RNA, or a chemically synthesized polynucleotide.
  • the polynucleotide can be cloned into an expression vector containing a constitutively active promoter segment (such as but not limited to CMV, SV40, Elongation Factor (EF) or LTR sequences) or to inducible promoter sequences such as the steroid inducible pIND vector (Invitrogen), tetracycline, or mouse mammary tumor virus (MMTV), where the expression of the dominant negative MMR gene can be regulated.
  • a constitutively active promoter segment such as but not limited to CMV, SV40, Elongation Factor (EF) or LTR sequences
  • inducible promoter sequences such as the steroid inducible pIND vector (Invitrogen), tetracycline, or mouse mammary tumor virus (MMTV)
  • the polynucleotide can be introduced into the cell by transfection.
  • a "promoter” is a DNA sequence that encompasses binding sites for trans -acting transcription factors. Promoters,
  • a targeting fragment containing 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 nts of 5' and 3' homologous sequence is transfected into MMR deficient cell hosts, the cell is grown and screened for clones containing chromosomes whereby the targeting fragment has been integrated.
  • MlVm defective cells may be of human, primates, mammals, rodent, fish, plant, fungal, yeast or of the prokaryotic kingdom.
  • Transfection is any process whereby a polynucleotide is introduced into a cell.
  • the process of transfection can be carried out in a living animal, e.g ., using a vector for gene therapy, or it can be carried out in vitro, e.g., using a suspension of one or more isolated cells in culture.
  • the cell can be any type of eukaryotic cell, including, for example, cells isolated from humans or other primates, mammals or other vertebrates, invertebrates, and single celled organisms such as protozoa, yeast, or bacteria.
  • transfection will be carried out using a suspension of cells, or a single cell, but other methods can also be applied as long as a sufficient fraction of the treated cells or tissue incorporates the polynucleotide so as to allow transfected cells to be grown and utilized.
  • Techniques for transfection are well known. Available techniques for introducing polynucleotides include but are not limited to electroporation ( Potter et al. (1988) Proc. Natl. Acad. Sci. USA 81:7161 ), transduction, cell fusion, the use of calcium chloride Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Press, New York, 2000 ) or calcium phosphate precipitation ( Wigler et al.
  • An isolated cell includes cells obtained from a tissue of humans, animals, plants or fungi by mechanically separating out individual cells and transferring them to a suitable cell culture medium, either with or without pretreatment of the tissue with enzymes, e.g ., collagenase or trypsin. Such isolated cells are typically cultured in the absence of other types of cells.
  • Cells selected for the introduction of a targeting fragment may be derived from a eukaryotic organism in the form of a primacy cell culture or an immortalized cell line, or may be derived from suspensions of single-celled organisms.
  • Integration of the targeting fragment can be detected by analyzing the chromosomal locus of interest for alterations in the genotype of the cells or whole organisms, for example by examining the sequence of genomic DNA, cDNA, RNA, or polypeptides associated with the gene of interest Integration can also be detected by screening for the expression levels of the targeted locus for altered expression profiles, or chimeric transcripts through biochemical methods or nucleic acid monitoring. Techniques for analyzing nucleic acids and proteins are well known in the art.
  • Techniques include, but are not limited to Southern analysis, northern analysis, PCR, reverse transcriptase-PCR (rt-PCR), restriction digest mapping, western blot, enzyme-linked immunosorbent assays (ELISA), radioimmunoassay, immunoprecipitation, and well-known variations of these techniques.
  • mismatch repair proteins that can be used for dominant negative MMR inhibitors and nucleic acid sequences include the following: human PMS2-134 protein (SEQ ID NO:11); human PNLS2-134 cDNA (SEQ ID NO:12).
  • the LSTFs for use in the invention may also be used to insert nucleic acid sequences through homologous recombination in cells that are naturally deficient in mismatch repair. Furthermore, cells may be rendered deficient in mismatch repair before, after or simultaneously with the introduction of the LSTFs.
  • EXAMPLE 1 Stable expression of dominant negative mismatch repair (MMR) genes in cells results in MMR inactivity.
  • MMR deficient cells Expression of a dominant negative allele in an otherwise mismatch repair (MMR) proficient cell can render these host cells MMR deficient ( Nicolaides, N.C. et al. (1998) Mol. Cell. Biol. 18:1635-1641 , U.S. Patent No. 6,146,894 to Nicolaides et al .).
  • the creation of MMR deficient cells can lead to the generation of genetic alterations throughout the entire genome of a host's offspring, yielding a population of genetically altered offspring or siblings that have an enhanced rate of homologous recombination. The generation of humans with germ line alterations is explicitly excluded.
  • This patent application teaches of the use of dominant negative MMR genes in cells, including but not limited to rodent, human, primate, yeast, insect, fish and prokaryotic cells with enhanced rates of homologous recombination followed by the introduction of locus specific targeting fragments (LSTFs) that can alter the expression of a chromosomal locus or integrate into a given exon of a gene for facilitated analysis of gene expression.
  • LSTFs locus specific targeting fragments
  • MI microsatellite instability
  • a method used to detect MMR deficiency in eukaryotic cells is to employ a reporter gene that has a polynucleotide repeat inserted within the coding region that disrupts its reading frame due to a frame shift.
  • the reporter gene will acquire random mutations (i.e . insertions and/or deletions) within the polynucleotide repeat yielding clones that contain a functional reporter gene.
  • pCAR-OF mammalian expression construct containing a defective ⁇ -galactosidase gene
  • the pCAR-OF vector consists of a ⁇ -galactosidase gene containing a 29-basepair poly-CA tract inserted at the 5' end of its coding region, which causes the wild-type reading frame to shift out-of-frame.
  • This chimeric gene is cloned into the pCEP4, which contains the constitutively cytomegalovirus (CMV) promoter upstream of the cloning site and also contains the hygromycin-resistance (HYG) gene that allows for selection of cells containing this vector.
  • CMV constitutively cytomegalovirus
  • HOG hygromycin-resistance
  • the pCAR-OF reporter cannot generate ⁇ -galactosidase activity unless a frame-restoring mutation (i.e., insertion or deletion) arises following transfection into a host.
  • Another reporter vector called pCAR-IF contains a ⁇ -galactosidase in which a 27-bp poly-CA repeat was cloned into the same site as the pCAR-OF gene, but it is biologically active because the removal of a single repeat restores the open reading frame and produces a functional chimeric ⁇ -galactosidase polypeptide (not shown).
  • 293PMS 134 and 293vec cells were transfected with the pCAR-OF reporter vector and selected for 17 days in neomycin plus hygromycin selection medium.
  • ⁇ -galactosidase expression of 293vec and 293PMS134134 cells transfected with pCAR-OF reporter vectors were transfected with the pCAR-OF ⁇ -galactosidase reporter plasmid. Transfected cells were selected in hygromycin and G418, expanded and stained with X-gal solution to measure for B-galactosidase activity (blue colored cells). 3 plates each were analyzed by microscopy. The results below represent the mean +/- standard deviation of these experiments.
  • 293PMS134/pCAR-OF clones that were pooled and expanded also showed a number of cells that contained a functional ⁇ -galactosidase gene. No ⁇ -galactosidase positive cells were observed in 293vec cells transfected with the pCAR-OF vector (data not shown). These data demonstrate the ability of dominant negative alleles of MMR genes to suppress endogenous MMR activity. These cells are now primed for the introduction of locus specific targeting fragments for altering the expression or tagging the exon of specific genes within the chromosomal context of the host.
  • 100,000 cells are harvested and fixed in 1% gluteraldehyde, washed in phosphate buffered saline solution and incubated in 1 ml of X-gal substrate solution (0.15 M NaCl, 1 mM MgCl 2 , 3.3 mM K 4 Fe(CN) 6 , 3.3 mM K 3 Fe(CN) 6 , 0.2% X-Gal ) in 24 well plates for 2 hours at 37°C. Reactions are stopped in 500 mM sodium bicarbonate solution and transferred to microscope slides for analysis. Three plates each are counted for blue ( ⁇ -galactosidase positive cells) or white ( ⁇ -galactosidase negative cells) to assess for MMR inactivation.
  • X-gal substrate solution 0.15 M NaCl, 1 mM MgCl 2 , 3.3 mM K 4 Fe(CN) 6 , 3.3 mM K 3 Fe(CN) 6 , 0.2% X-Gal
  • EXAMPLE 2 Generation of targeting cassettes for altered gene expression or tagged exons for expression profiling of host organisms.
  • MMR defective cells have a higher rate of homologous recombination due to the decreased stringency for identical basepair matches of the target vector to the chromosomal locus.
  • LSTFs containing short areas of homology for rapid genome targeting of chromosomal loci we employed the use of MMR defective 293 cells (293PMS134) that express the PMS134 dominant negative allele as described in Example 1.
  • MMR defective 293 cells (293PMS134) that express the PMS134 dominant negative allele as described in Example 1.
  • CMV Cytomegalovirus
  • PCR products were amplified from the p4 plasmid, which contains a DNA insert with the Thymidine Kinase (Tk) promoter upstream of the hygromycin resistance (Hyg) gene followed by the SV40 polyadenylation signal and the cytomegalovirus (CMV) promoter. Plasmid was amplified with primers containing 3' sequences that are homologous to the plasmid vector sequence region upstream of the Tk promoter and downstream of the CMV promoter. Each primer also contained 70 nt that were homologous to the genomic locus of various target genes at the start site of transcription. PCRs were typically carried out using buffers as previously described ( Grasso, L. et al.
  • HEK293 human embryonic kidney cells stably expressing the PMS134 gene (see Example 1) were transfected with 1 ⁇ g of purified PCR products from above using 3 ⁇ l Fugene6 (Invitrogen) and stable transfectant pools were generated by co-selection with 100 ⁇ g/ml hygromycin B and G418 (neomycin). Cultures were selected for 14 days in neomycin and hygromycin. Pools and clones were analyzed for locus specific integration using reverse transcriptase coupled PCR as described ( Nicolaides, N.C. et al.
  • the target genes were the human N-Ras (a signal transduction gene), beta-globin (a structural protein), INF-gamma (a secreted growth factor), and galanin receptor (a seven transmembrane G-coupled receptor).
  • each primer used for each 5' flanking locus is given below in Table 2 where the last 30 nts of each primer is specific for the 5' and 3' ends of the targeting fragment containing the Tk promoter driving hygromycin expression followed by the CMV promoter, while the 5' ends of each primer pair are specific to the 5'flanking region of each locus, N-RAS (SEQ ID NO: 13 and 14); beta-globin (SEQ ID NO: 15 and 16); Interferon gamma (SEQ ID NO: 17 and 18); and galanin receptor (SEQ ID NO: 19 and 20).
  • Transfected cells were first analyzed by RT-PCR analysis to identify increased steady-state gene expression using primer pairs that were capable of detecting spliced mRNA (primers listed in Table 3). These primer combinations can detect the endogenous gene expression of a target gene independent of LSTF integration. Expression analysis of transfected cells failed to reveal robust expression levels of any of these four loci in parental HEK293 or control HEK293 cells transfected with the different fragments. Conversely, robust expression was observed for all targeted loci in transfected 293PMS134 cells containing the appropriate LSTF. A representative example is shown using cells where the beta-globin locus was targeted.
  • HEK293 cells which are derived from embryonic kidney have not been found to express the erythroid-specific beta-globin.
  • Shown in Figure 2 is expression analysis of beta-globin using cDNA specific primers (SEQ ID NO: 24 and SEQ ID NO:25, Table 3) in targeted cells containing the beta-globin LSTF, while none was observed in cells transfected with targeting vectors to other loci, which served as negative controls.
  • An independent RT-PCR was carried using cDNA from the positive cultures using a 5' primer that was located in the distal leader sequence of he CMV promoter (SEQ ID NO:21, Table 3) and a 3' primer located within the coding region of the beta-globin gene (SEQ ID NO: 25, Table 3).
  • This primer set is only capable of producing a product with an expected molecular weight if the LSTF is integrated within the specific targeted locus because the resultant product consists of a hybrid transcript consisting of a cDNA comprised of a CMV leader fused to the initiating start codon for the targeted gene, which can only occur by correct genome integration for formation of this hybrid message. Similar results were found using targeting fragments to other chromosomal loci as well as using primers containing 50 nts of flanking sequence, whereas no locus specific expression was observed in HEK293 control cells transfected with similar fragments (data not shown). Table 2. Transfection construct primers.
  • Analysis of cell lines transfected with promoter-specific LSTFs can be carried out by any number of methods that measure levels of RNA or proteins. Such methods of analysis may include but are not limited to microarray analysis, in situ RT-PCR, Northern blot, western blotting, immunostaining, fluorescent Activated Cell Sorting, etc . Cell lines over expressing a gene of interest may be analyzed by functional assays using biological systems that are sensitive to the production of certain biochemicals of growth factors. These methods are routinely used by those skilled in the art of high throughput screening and are useful for analyzing the expression levels of target genes in cells transfected with LSTFs.
  • LST exon locus specific targeting
  • the LST fragment contains a selectable marker fused to a reporter gene that can be used in combination with any number of analytical systems to monitor gene expression in situ or in vitro.
  • An example of one such fusion cassette is presented in Figure 3 , whereby the hygromycin resistance gene is fused in-frame with the luciferase gene.
  • fusion expression cassettes that contain a selectable maker fused in-frame with a reporter gene.
  • Exon LSTFs is generated by PCR using 80-100 nt primers that contain 50-70 nts of 5' sequence that are homologous to the 5' and 3' boarders of a given gene's exon, while the terminal 30nts are specific for the first and last codons of the fusion protein, such as those given as examples in Figure 3 .
  • PCR products are amplified from the pFusion plasmid, containing a DNA insert with the selectable marker/reporter gene. PCRs are carried out using buffers as previously described ( Grasso, L. et al. (1998) "Molecular analysis of human interleukin-9 receptor transcripts in peripheral blood mononuclear cells.
  • EXAMPLE 3 Generation of targeting cassettes for altered gene expression or tagged chromosomes for site-specific gene amplification.
  • Another means for enhancing gene expression from the genome of a host organism is through the process of gene amplification.
  • a number of studies have reported the use of expression vectors consisting of a gene of interest linked to a DHFR expression cassette. Once the expression vector has been inserted into the genome of a host cell line, expression cassettes can be amplified by selecting for clonal resistance to methotrexate, a process that occurs through gene amplification of the DHFR gene and surrounding proximal and distal loci ( Ma, C. et al. (1993) "Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells" Genes Dev. 7:605-620 ).
  • a method is taught here that employs the use of LSTFs in MMR defective cells via the use of MMR inhibitors, whereby the LSTF contains a constitutively expressed DHFR gene juxtaposed to selectable markers with the ends of the LSTF containing 50-70 bps of homologous sequence to an endogenous gene locus.
  • the target site may be proximal, intragenic or distal to the target locus.
  • the LSTF is generated from a Hyg-DHFR cassette via PCR using the pHYG-DHFR vector as template.
  • Amplifications are generated using primers that are 5' to the TK promoter, which controls the HYG expression and a primer that is directed to the sequence 3' of the DHFR gene, which consists of the SV40polyA signal.
  • Each primer contains 50-70 nts that are homologous to the chromosomal target site.
  • Cells are transfected with a dominant negative MMR expression vector, which contains a neomycin resistance marker as described in Example 1 along with the LSTF. Upon cotransfection, cells are coselected in hygromycin and neomycin for 14 days. Cells are analyzed for chromosomal specific integration using primers that flank the targeted site of integration. Analysis can be in pooled cultures or in single clones.
  • cells are selected for chromosomal site-specific amplification by methotrexate (MTX) selection. Briefly, 1.0 x 10 6 cells are seeded in 10cm culture dishes with complete growth medium supplemented with 10% dialyzed fetal bovine serum 24 h prior to drug selection. Next, MTX is added at 15 times the calculated IC 50 and the plates are incubated at 37°C. Cells are grown in the presence of continuous MTX selection for 14 to 21 days. Colonies are selected and analyzed for DHFR and chromosome amplification. Analysis of genomic DNA is carried out using the modified salting out method. Briefly, cells are isolated from parental or MTX exposed clones.
  • MTX methotrexate
  • Cells are pelleted and lysed in 1 ml of lysis buffer (25 mM Tris-HCl pH 8.0, 25 mM EDTA, 1% SDS, 0.5 mg/ml proteinase K). Cell lysates are incubated at 50 °C 12 hrs to overnight. Following ethanol precipitation and resuspension, RNaseA was added to 100 ⁇ g/ml and the mixture was kept at 37°C for 30 min. Next, DNAs are phenol extracted and precipitated by the addition of 3 M NaOAc and ethanol. DNA pellets are washed once with 70% ethanol, air-dried and resuspended in TE buffer. DNAs are digested with different restriction enzymes and probed for DHFR and the locus of interest for amplification as compared to the control cells. MMR activity is restored in amplified clones and the cells are used for experimentation or production.
  • lysis buffer 25 mM Tris-HCl pH 8.0, 25 mM EDTA
  • a benefit taught by this application is the combined use of MMR deficiency, enhanced homologous recombination with LSTFs and the ability to produce site-specific gene amplification within a host's genomic locus.
  • the blockade of MMR in cells to increase LSTF integration can be through the use of dominant negative MMR gene alleles from any species including bacteria, yeast, protozoa, insects, rodents, primates, mammalian cells, and man.
  • Blockade of MMR can also be generated through the use of antisense RNA or deoxynucleotides directed to any of the genes involved in the MMR biochemical pathway.
  • Blockade of MMR can be through the use of polypeptides that interfere with subunits of the MMR complex including but not limited to antibodies.
  • the blockade of MMR may be through the use of chemicals such as but not limited tononhydrolyzableATP analogs, which have been shown to block MMR ( Galio, L. et al.

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Claims (13)

  1. Procédé in vitro pour introduire un fragment de ciblage à spécificité de locus dans le génome d'une cellule par recombinaison homologue, consistant à :
    inhiber la réparation de mésappariement endogène de ladite cellule en introduisant dans ladite cellule un allèle négatif dominant d'un gène PMS2 constitué de PMS2-134 ; et
    introduire un fragment de ciblage à spécificité de locus dans ladite cellule
    dans lequel ledit fragment de ciblage à spécificité de locus est un polynucléotide comprenant au moins un promoteur, un marqueur sélectionnable et des régions flanquantes en 5' et 3' ayant de 20 à 120 nucléotides ; dans lequel lesdites régions flanquantes en 5' et 3' sont homologues d'une portion sélectionnée du génome de ladite cellule ; et dans lequel ledit fragment de ciblage à spécificité de locus s'intègre dans le génome de ladite cellule par recombinaison homologues.
  2. Procédé in vitro pour altérer génétiquement une cellule afin qu'elle surproduise un polypeptide sélectionné, consistant à :
    inhiber la réparation de mésappariement endogène de ladite cellule en introduisant dans ladite cellule un allèle négatif dominant d'un gène PMS2 constitué de PMS2-134 ;
    introduire un fragment de ciblage à spécificité de locus dans ladite cellule
    dans lequel ledit fragment de ciblage à spécificité de locus est un polynucléotide comprenant au moins une séquence de promoteur, un marqueur sélectionnable et des régions flanquantes en 5' et 3' ayant de 20 à 120 nucléotides, dans lequel lesdites régions Manquantes en 5' et 3' sont homologues d'une portion sélectionnée du génome de ladite cellule, et dans lequel ledit fragment de ciblage à spécificité de locus intègre dans le génome de ladite cellule par recombinaison homologue,
    dans lequel l'intégration dudit fragment de ciblage à spécificité de locus augmente l'expression d'un locus de gène souhaite ; et
    sélectionner ladite cellule qui surproduit ledit polypeptide sélectionné.
  3. Procédé in vitro pour étiqueter un exon d'une cellule afin de cribler l'expression d'un gène en réponse à des composés biochimiques ou pharmaceutiques, consistant à :
    inhiber la réparation de mésappariement endogène de ladite cellule en introduisant dans ladite cellule un allèle négatif dominant d'un gène PMS2 constitué de PMS2-134 ; et
    introduire un fragment de ciblage à spécificité de locus dans ladite cellule
    dans lequel ledit fragment de ciblage à spécificité de locus est un polynucléotide comprenant un élément rapporteur, un marqueur sélectionnable et des régions flanquantes en 5' et 3' ayant de 20 à 120 nucléotides, dans lequel lesdites régions flanquantes en 5' et 3' sont homologues d'une portion sélectionnée du génome de ladite cellule ; dans lequel ledit fragment de ciblage à spécificité de locus s'intègre à l'intérieur d'un exon de gène ciblé par recombinaison homologué ; et dans lequel lesdites cellules contenant des gènes avec des exons étiquetés sont utilisées pour le criblage de l'expression d'un gène en réponse à des composés biochimiques ou pharmaceutiques.
  4. Procédé selon la revendication 3, dans lequel ledit élément rapporteur est choisi dans le groupe constitué par la luciférase et la protéine fluorescente verte.
  5. Procédé selon la revendication 3 ou la revendication 4, dans lequel ledit élément rapporteur est fusionné dans le cadre audit marqueur sélectionnable.
  6. Procédé in vitro pour étiqueter un site chromosomique spécifique pour une amplification de gène à spécificité de locus, consistant à :
    inhiber la réparation de mésappariement endogène de ladite cellule en introduisant dans ladite cellule un allèle négatif dominant d'un gène PMS2 constitué de PMS2-134 ; et
    introduire un fragment de ciblage à spécificité de locus dans ladite cellule
    dans lequel ledit fragment de ciblage à spécificité de locus est un polynucléotide comprenant, liés de manière fonctionnelle un gène de dihydrofolate réductase, un promoteur, et des régions flanquantes en 5' et 3' ayant de 20 à 120 nucléotides, dans lequel lesdites régions flanquantes en 5' et 3' sont homologues d'une portion sélectionnée du génome de ladite cellule ; dans lequel ledit fragment de ciblage à spécificité de locus s'intègre dans le génome de ladite cellule par recombinaison homologue ; et dans lequel ledit site chromosomique spécifique est étiqueté pour une amplification de gène à spécificité de locus.
  7. Procédé selon la revendication 6, dans lequel ledit fragment de ciblage à spécificité de locus comprend en outre un marqueur sélectionnable et un deuxième promoteur lié de manière fonctionnelle audit marqueur sélectionnable.
  8. Procédé selon l'une quelconque des revendications précédentes, comprenant en outre la restauration de l'activité de réparation de mésappariement de ladite cellule,
  9. Procédé selon l'une quelconque des revendications 1, 2 et 6, dans lequel ledit promoteur est choisi dans le groupe constitué par un promoteur CMV, un promoteur SV40, un facteur d'allongement, une séquence LTR, une séquence de promoteur pIND, une séquence de promoteur de tétracycline, et une séquence de promoteur MMTV.
  10. Procédé selon l'une quelconque des revendications 1, 2 et 3, dans lequel ledit marqueur sélectionnable est choisi dans le groupe constitué par un gène de résistance à l'hygromycine, un gène de résistance à la néomycine et un gène de résistance à la zéocine,
  11. Procédé selon l'une quelconque des revendications précédentes, dans lequel lesdites régions flanquantes en 5' et 3' ont une longueur de 30 à 100 nucléotides, une longueur de 40 à 90 nucléotides, une longueur de 50 à 80 nucléotides ou une longueur de 50 à 70 nucléotides.
  12. Procédé selon l'une quelconque des revendications précédentes, dans lequel ladite cellule est choisie dans le groupe constitué par une cellule de vertébré, une cellule d'invertébré, une cellule de mammifère, une cellule de reptile, une cellule fongique, et une cellule de levure,
  13. Procédé selon l'une quelconque des revendications précédentes, dans lequel lesdites régions flanquantes en 5' et 3' sont homologues d'une région flanquante en 5' d'un locus chromosomique sélectionné de ladite cellule.
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EP1268765B1 (fr) * 2000-02-11 2008-08-13 The Johns Hopkins University Procedes d'obtention de microbes hypermutables
EP1282708B1 (fr) 2000-02-18 2008-07-02 Morphotek, Inc. Procede de generation de plantes tres sujettes aux mutations
CA2399191C (fr) * 2000-02-23 2010-12-14 The Johns Hopkins University Procedes de generation de levures presentant une hyper mutabilite
US6569681B1 (en) * 2000-03-14 2003-05-27 Transkaryotic Therapies, Inc. Methods of improving homologous recombination
US6596541B2 (en) * 2000-10-31 2003-07-22 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
DE60144247D1 (de) 2001-01-15 2011-04-28 Morphotek Inc Chemische hemmstoffe des mismatch-repair
EP1572935A4 (fr) 2002-02-21 2006-09-13 Morphotek Inc Procedes de fabrication de cellules hypermutables utilisant des homologues de pmsr

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CA2473741C (fr) 2015-12-22
US7638334B2 (en) 2009-12-29
WO2003062435A1 (fr) 2003-07-31
EP1474522A4 (fr) 2005-09-07
AU2003205173B2 (en) 2008-05-22
CA2473741A1 (fr) 2003-07-31
US20030176386A1 (en) 2003-09-18
EP1474522A1 (fr) 2004-11-10
AU2003205173C1 (en) 2009-01-29
ATE548461T1 (de) 2012-03-15

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